Lead(II) as a probe for investigating RNA structure in vivo
Lead(II) as a probe for investigating RNA structure in vivo
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DOI：
10.1017/s1355838201020416
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发表时间：
2002-04-01
期刊：
影响因子：
4.5
通讯作者：
Wagner, EGH
中科院分区：
文献类型：
--
作者：
Lindell, M;Romby, P;Wagner, EGH
在这篇通讯中，我们描述了一种简单可靠的RNA结构测定方法，在体内，使用二价狮子，铅（II）作为结构探针。已知铅（II）可以在单链区域、环和凸起处切割RNA，而在双链区域的切割较弱或不存在。由于离子容易进入细菌细胞，因此在大肠杆菌培养物中加入50-100 mM的醋酸铅（II）处理3-7分钟，导致RNA在体内部分切割。通过对提取的总RNA进行逆转录分析，确定了切割位置。分析了三种RNA: tmRNA， CopT（反义RNA CopA的靶标）和ompF mRNA的先导区。这三种rna先前已经在体外进行了分析，并且二级结构模型可用。本文的研究结果表明，体内的铅（11）切割产生了这些rna的详细结构信息，这与基于体外工作提出的模型很好地一致。这些数据说明了铅（II）作为一种序列无关的RNA结构探针在活细胞中的应用潜力。
In this communication, we describe a simple and reliable method for RNA structure determination In vivo, using the divalent lion, lead(II), as a structural probe. Lead(II) is known to cleave RNA within single-stranded regions, loops, and bulges, whereas cleavages in double-stranded regions are weaker or absent. Because the Ion easily entered bacterial cells, Escherichia coli cultures were treated by addition of 50-100 mM lead(II) acetate for 3-7 min, resulting in partial cleavage of RNA in vivo. Cleavage positions were mapped by reverse transcription analysis of total extracted RNA. Three RNAs were analyzed: tmRNA, CopT (the target of the antisense RNA CopA), and the leader region of the ompF mRNA. All three RNAs had previously been analyzed in vitro, and secondary structure models were available. The results presented here show that lead (11) cleavages in vivo yield detailed structural information for these RNAs, which was in good agreement with the models proposed based on in vitro work. These data illustrate the potential of lead(II) as a sequence-independent RNA structure probe for use in living cells.